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Production of high affinity monoclonal antibodies to deoxycorticosterone

By Emad A S Al-Dujaili, A Hubbard, V van Heyningen and C Edwards

Abstract

Monoclonal antibodies to deoxycorticosterone were produced. Mice were immunised with deoxycorticosterone-3-mono-oxime-BSA conjugate and spleen cells were then hybridised with NS1/1Ag4-1 mouse myeloma cells using 1500 mol. wt polyethylene glycol. The hybrids were grown in RPMI 1640 medium containing HAT to facilitate selection of positive clones. The clones and subclones were screened by using deoxycorticosterone-3-mono-oxime-[125I]iodohistamine. Dextran-coated charcoal was used for separation of antibody bound and free fractions. Two independent clones producing antibody which specifically binds labelled deoxycorticosterone were obtained. Cells from the two best sub-clones were used to raise ascites fluid. Comparison of these antibodies with one of the best conventional antisera previously raised in rabbits showed that the affinity constants were almost comparable (0.49-1.4 X 10(10) l/mol). Cross-reactivity of monoclonal antibodies with cortisol, corticosterone, testosterone and pregnenolone was lower than for polyclonal antisera, but for progesterone the cross-reactivity was similar in both cases. The assay sensitivity obtained with ascites fluid was comparable to that of conventional antibody (2.5 pg/ml). The dilution of ascites fluid which produced 50% binding of the label was 1:4,000,000. These results confirm that it is possible to produce monoclonal antibodies to corticosteroids

Publisher: elsivier
OAI identifier: oai:eresearch.qmu.ac.uk:1177
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